DETECTION OF NUCLEIC ACID SEQUENCE DIFFERENCES USING COUPLED LIGASE DETECTION AND POLYMERASE CHAIN REACTIONS

• US 20120252696A1
• Filed: 06/14/2012
• Published: 10/04/2012
• Est. Priority Date: 05/29/1996
• Status: Active Grant

• Current Assignee: Cornell Research Foundation Incorporated (Cornell University)
• Original Assignee: Cornell Research Foundation Incorporated (Cornell University)
• Inventors: BARANY, Francis, LUBIN, Matthew, BARANY, George, HAMMER, Robert P.
• Granted Patent: US 8,597,890 B2
• Time in Patent Office: Days
• US Class Current: 506/9
• CPC Class Codes: C12Q 1/6813 Hybridisation assays
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/683 involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
• CPC Class Codes: C12Q 1/6851 Quantitative amplification
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6862 Ligase chain reaction [LCR]
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 1/6881 for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
• CPC Class Codes: C12Q 2521/319 Exonuclease
• CPC Class Codes: C12Q 2521/501 Ligase
• CPC Class Codes: C12Q 2521/531 Glycosylase
• CPC Class Codes: C12Q 2525/125 incorporating agents resulting in resistance to degradation
• CPC Class Codes: C12Q 2525/131 incorporating a restriction site
• CPC Class Codes: C12Q 2525/155 incorporating/generating a new priming site
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Q 2533/107 Probe or oligonucleotide ligation
• CPC Class Codes: C12Q 2535/131 : Allele specific probes
• CPC Class Codes: C12Q 2537/143 : Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2545/114 : involving a quantitation step
• CPC Class Codes: C12Q 2561/125 : Ligase Detection Reaction [LDR]
• CPC Class Codes: C12Q 2600/156 : Polymorphic or mutational markers
• CPC Class Codes: C12Q 2600/16 : Primer sets for multiplex assays

Abstract

The present invention relates to a method for identifying a target nucleotide sequence. This method involves forming a ligation product on a target nucleotide sequence in a ligation detection reaction mixture, amplifying the ligation product to form an amplified ligation product in a polymerase chain reaction (PCR) mixture, detecting the amplified ligation product, and identifying the target nucleotide sequence. Such coupling of the ligase detection reaction and the polymerase chain reaction permits multiplex detection of nucleic acid sequence difference.

Claims

1. A method for identifying one or more of a plurality of sequences differing by one or more single-base changes, insertions, deletions, or translocations in a plurality of target nucleotide sequences comprising:

subjecting a reaction mixture to a ligation process to produce one or more ligation products, wherein the reaction mixture comprises;

a ligase;

one or more target nucleotide sequences; and

one or more oligonucleotide probe sets, each probe set comprising (a) a first oligonucleotide probe comprising a first target-specific portion capable of hybridizing to a first portion of a corresponding target nucleotide sequence and (b) a second oligonucleotide probe comprising a second target-specific portion capable of hybridizing to a second portion of a corresponding target nucleotide sequence, wherein each of the one or more ligation products comprises a ligated sequence which includes (1) the first target-specific portion of the first oligonucleotide probe in a corresponding probe set and (2) the second target-specific portion of the second oligonucleotide probe in the corresponding probe set;

subjecting the one or more ligation products to one or more polymerase chain reaction cycles to produce one or more extension products comprising the ligated sequences and/or complements thereof; and

detecting the extension products to identify one or more target nucleotide sequences in the sample.

2. The method according to claim 1, wherein the first oligonucleotide probe comprises a 5′

upstream primer-specific portion and a further portion and the second oligonucleotide probe comprises a 3′

downstream primer-specific portion, wherein the ligated sequence of each of the ligation products comprises the 5′

upstream primer-specific portion and a further portion and the 3′

downstream primer-specific portion.

3. The method according to claim 2 further comprising:

providing one or a plurality of oligonucleotide primer sets, each primer set comprising (a) an upstream primer and (b) a downstream primer, wherein in each primer set, one of said upstream or downstream primers comprises a detectable reporter label.

4. The method according to claim 3, wherein the detectable reporter label is selected from the group consisting of chromophores, fluorescent moieties, enzymes, antigens, heavy metals, magnetic probes, dyes, phosphorescent groups, radioactive materials, chemiluminescent moieties, and electrochemical detecting moieties.

5. The method according to claim 3, wherein the upstream primer contains the same sequence as the 5′

upstream primer-specific portion and the downstream primer contains a sequence complementary to the 3′

downstream primer-specific portion.

6. The method according to claim 2 further comprising:

providing a solid support with different capture oligonucleotides immobilized at different particular sites on the solid support, wherein the capture oligonucleotides have nucleotide sequences complementary to the further portions; and

contacting the extension products with the solid support under conditions effective to hybridize the further portions of the extension products to the capture oligonucleotides in a base-specific manner, thereby capturing the extension products to the solid support at the particular site with the complementary capture oligonucleotide, wherein said detecting comprises detecting the extension products captured to the solid support due to hybridization between the corresponding further portions and the complementary capture oligonucleotides at the particular sites.

7. The method according to claim 6, wherein the further portion is an addressable array portion.

8. The method according to claim 1, wherein the target-specific portions of the oligonucleotide probes each have a hybridization temperature of 50-85°

C.

9. The method according to claim 1, wherein the target-specific portions of the oligonucleotide probes are 20-28 nucleotides long.

10. The method according to claim 1, wherein the oligonucleotide probe sets are selected from the group consisting of ribonucleotides, deoxyribonucleotides, modified ribonucleotides, modified deoxyribonucleotides, modified phosphate-sugar backbone oligonucleotides, nucleotide analogues, and mixtures thereof.

11. The method according to claim 1, wherein the ligase is selected from the group consisting of a Thermus aquaticus ligase, a Thermus thermophilus ligase, an E. coli ligase, T4 ligase, and a Pyrococcus ligase.